Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors.

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 µM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 µM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

Development of a G118R mutation in HIV-1 integrase following a switch to dolutegravir monotherapy leading to cross-resistance to integrase inhibitors.

OBJECTIVES: Dolutegravir shows a high barrier to resistance with no previously reported cases of acquired integrase mutations during first-line therapy. In this study, rapid development of the G118R mutation arose following a switch from first-line elvitegravir/cobicistat/tenofovir disoproxil fumarate/emtricitabine to dolutegravir monotherapy. The G118R mutation also arose in a treatment-experienced patient switched to dolutegravir monotherapy. The genetic basis for G118R selection and potential phenotypic outcome was ascertained. PATIENT AND METHODS: Genotypic analysis was performed on patients with virological failure (<1000 copies/mL) on dolutegravir-containing regimens. The Los Alamos database was queried for glycine codon 118 polymorphisms. Cell culture selections and phenotypic drug susceptibility assays assessed resistance via the G118R pathway. RESULTS: We report on two patients who developed viral failure while on dolutegravir monotherapy. Both patients had been on a current or previous regimen containing integrase inhibitors. Virological failure (<1000 copies/mL) emerged early within 2 months following the dolutegravir switch. The appearance of G118R in these two cases and subtype C and CRF02_AG in vitro selections were related to a rare GGA natural polymorphism at codon 118 (1.5% prevalence), facilitating a GGA to AGA transition. Cell culture selections were used to assess the in vitro progression of the G118R pathway leading to cross-resistance to all integrase inhibitors. CONCLUSIONS: Although resistance to dolutegravir is typically rare, genetic polymorphisms and monotherapy can facilitate the acquisition of G118R.

Loss of Runx3 function in leukocytes is associated with spontaneously developed colitis and gastric mucosal hyperplasia.

RUNX transcription factors are key regulators of lineage-specific gene expression and might be involved in autoimmune diseases. Runx3 plays a role during the development of sensory neurons and T cells and regulates transforming growth factor beta (TGF-beta) signaling in dendritic cells. Here, we report that at 4 weeks of age, Runx3 knockout (KO) mice spontaneously develop inflammatory bowel disease (IBD) characterized by leukocyte infiltration, mucosal hyperplasia, formation of lymphoid clusters, and increased production of IgA. Additionally, at a considerably older age (8 months), the KO mice also develop progressive hyperplasia of the gastric mucosa associated with disturbed epithelial differentiation and cellular hyaline degeneration. Analysis of cytokines in the colonic mucosa of Runx3 KO mice revealed a mixed T helper 1/T helper 2 response. By using immunohistochemistry and RNA in situ hybridization, Runx3 expression in the gastrointestinal tract is detected in lymphoid and myeloid populations but not in the epithelium. The data indicate that loss of leukocytic cell-autonomous function of Runx3 results in IBD and gastric lesion in the KO mice. IBD in humans is viewed as a complex genetic disorder. Several susceptibility loci were identified on different human chromosomes including the chromosomal region 1p36 where RUNX3 resides. It is thus tempting to speculate that mutations in RUNX3 may constitute an IBD risk factor in humans.